Flow cytometry has become an indispensable tool in clinical and basic immunological research due to its ability to distinguish subsets in heterogeneous populations of cells. Recently, major advances have been made in both flow cytometry instrumentation and applications, expanding the number of possible simultaneous analysis parameters to thirteen or more. With more parameters available, researchers have begun to identify more well-defined and biologically interesting subsets of lymphocytes samples based upon surface epitope staining.
Flow cytometry is routinely used for the identification of cellular populations based on a surface phenotype and also used for cellular based assays such as cytotoxicity, viability, and apoptosis, among others. It is well understood that flow cytometry offers the capability to assess the heterogeneity of cellular subsets that exist in complex populations such as peripheral blood. Although surface staining may be an effective means of characterizing cells, it does not provide information about the functional responses of those cells to stimuli that are immediately reflective of intracellular events. Even in cases where the marker used is a cytokine receptor or receptor tyrosine kinase, levels of the antigen do not always correlate with cellular response to the specific ligand. Therefore, methods have been developed to characterize cells by measuring levels of intracellular epitopes: cytokines, DNA, mRNA, enzymes, hormone receptors, cell cycle proteins, and of phosphorylated signaling molecules. As a result, research applications of flow cytometry are being increasingly applied to the measurement of intracellular proteins that regulate cell processes and represent important therapeutic targets for novel anticancer agents.
Current proteomic approaches, such as 2-dimensional SDS-PAGE and Mass-Spectroscopy of protein post-translational modifications are extremely powerful and have provided valuable insights into many intracellular activation processes. However, as the cells are lysed, it is obvious that the readout of these experiments is an average for protein activation states across the cell population(s). Significant biological events can be masked by such averaging, as there is no provision for the collection of information on the distribution of protein activation in individual cells within a population nor is there the ability to retroactively identify the cellular populations that corresponded to the detectable levels of active proteins. Therefore significant information on immune cell population variations that exist in both defined cellular populations and across different cell subtypes is undetectable and cannot be addressed by methodologies that require cell lysis for protein analysis. Ultimately, protein activation signaling cascades must be measured in their biological context to be both relevant and free of artifact.
Of particular interest has been the recent development of techniques for the analysis of signal transduction pathways, based on the use of phosphorylation state-specific antibodies. Multiparameter flow cytometric analysis allows for small subpopulations—representing different cellular subsets, differentiation or activation states—to be discerned using cell surface markers. As such, the usage of single cell techniques to characterize signaling events provides the ability to perform multiparametric experiments to identify the distinct signaling junctures of particular molecules in defined lymphocyte populations and to obtain a global understanding of the extent of signaling networks by correlating several active kinases involved in signaling cascades simultaneously, at the single cell level. Furthermore, the incorporation of these methods into conventional clinical flow cytometry protocols will have far-reaching application for the classification of hematological malignancies including the selection of patients for highly specific molecular cancer therapeutics, and for monitoring drug effects in patients.
Analysis of signal transduction pathways by flow cytometry presents technical problems that are not currently encountered in routine clinical applications. The phosphorylation states of individual signaling elements are rapidly modified in response to specific kinases and phosphatases, and therefore subject to artifactual changes during sample storage and preparation. Cellular responses to activating or inhibitory inputs are likely to be more informative than steady state measurements of phosphorylation states. Many anticancer agents show reversible binding to their molecular target. As a result, pharmacodynamic monitoring has to measure whole blood samples rather than isolated leukocytes. Ultimately, the existing and potential applications of phospho-specific flow cytometry to clinical settings, including characterization of immune system development and signaling, antigen-specific T-cell responses, drug screening, and disease phenotyping, must take into account that phosphorylation is a transient, reversible event that is indicative of the activation status of signaling proteins.
Thus, there exists a need to develop methods capable of biological sample preparation for signal transduction measurements by flow cytometry that are robust and suitable for general clinical application and able to capture the phosphorylation events that represent the activation status of signaling proteins. The present invention satisfies this need and provides related advantages as well.